Visualizing and Elucidating the Role of Force on Type IV Collagen in Development

Duke University, Durham NC

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Abstract

Visualizing and elucidating the role of force on type IV collagen in development PROJECT SUMMARY Basement membranes are highly conserved, dense, sheet-like extracellular matrices that surround most tissues and organs. Basement membranes provide mechanical strength to developing tissues, and loss or mutations in basement membrane components results in embryonic lethality, developmental defects, and numerous human diseases. Type IV collagen has been proposed to be the key structural component in basement membranes that provides mechanical stability, and mutations in collagen result in devastating developmental disorders that affect multiple dynamically growing and mechanically active tissues, including the vasculature, muscles, and brain. Owing to the difficulty of visualizing and experimentally examining type IV collagen dynamics and basement membrane components in complex vertebrate tissues in vivo, however, it is unknown how type IV collagen is assembled in basement membranes and whether it directly bears load. We have made C. elegans strains expressing functional GFP, Venus, and mCherry tagged versions of collagen and most other basement membrane proteins and receptors. We have also developed a photoconvertible Dendra-tagged collagen strain to optical highlight and track collagen deposited in basement membrane. C. elegans encodes all major basement membrane components with only a single gene representing each family, making it a powerful experimental model to dissect type IV collagen function and basement membrane regulation in vivo. The C. elegans pharynx is encased in a BM and is a rapidly growing contractile organ that initiates pumping in the embryo. The posterior terminal pharyngeal bulb is the site of food grinding, a region of high mechanical activity. The C. elegans pharynx is first covered with basement membrane during early embryogenesis, prior to pharyngeal pumping. We have found that type IV collagen is initially localized uniformly around the developing pharynx in the embryo, but after the pharynx initiates pumping, becomes enriched specifically around the terminal bulb. Loss of type IV collagen leads to pharyngeal pumping and morphological defects, indicating a critical role for collagen in pharyngeal development and function. The goal of this proposal is to combine live-cell imaging of type IV collagen with genetic analysis, RNAi knockdown, and force manipulations and development of a new collagen Fluorescence Energy Transfer (FRET)-based force sensor to: (1) Elucidate the biochemical and biophysical mechanisms of collagen addition to the basement membrane of the growing pharynx and the role of mechanical force in collagen recruitment; and (2) Visualize the load on type IV collagen in situ and determine if collagen is preferentially recruited to BM in areas of high mechanical activity. These studies are relevant to NIH's mission as they will lead to new mechanistic insights into the function, regulation, and assembly of type IV collagen in BMs, thus allowing a better understanding of the basis of human developmental disorders that result from defects in type IV collagen.